Vpliv različic v genu EGLN1 na stabilnost proteinskega produkta

Avguštinčič, Nuša (author), Debeljak, Nataša (mentor)

Eritrocitoza (ECYT) je bolezensko stanje, pri katerem pride do povečanega števila eritrocitov in posledično višje koncentracije hemoglobina v krvi. Pomembno vlogo pri uravnavanju eritropoeze ima s hipoksijo inducirani transkripcijski dejavnik (HIF). Njegovo stabilnosti in aktivnost uravnava prolil hidroksilaza 2 (PHD2), ki je kodirana z genom EGLN1. Nekatere genetske različice EGLN1 vplivajo na zmanjšano stabilnost EGLN1/PHD2 in so posledično povezane s pojavom eritrocitoze. Namen magistrske naloge je bil preučiti vpliv dveh novih genetskih različic v genu EGLN1, c.1072C>T, p.(Pro358Ser) in c.1124A>G, p.(Glu375Gly), na stabilnost proteina PHD2. Stabilnost proteinov različic EGLN1/PHD2 (p.(Glu375Gly) in p.(Pro358Ser)) smo primerjali z divjim tipom (WT) ter s pozitivno (p.(His374Arg)) in negativno (p.(Gln157His) kontrolo. V celično linijo HEK293 smo vstavili EGLN1 z izbranima različicama in kontrolami, po 24h smo translacijo zaustavili z dodatkom cikloheksimida ter celice lizirali v časovnih točkah 0-10 ur po dodatku cikloheksimida (T0, T3, T6, T10). Uspešnost translacije smo preverili z analizo prenosa po westernu. Rezultati so pokazali, da WT in negativna kontrola ne vplivata na proteinsko stabilnost EGLN1/PHD2, medtem ko ostale različice vplivajo na zmanjšano stabilnost proteina EGLN1/PHD2. Za različici p.(His374Arg) in p.(Pro358Ser) smo dobili statistično značilne rezultate, medtem ko za p.(Glu375Gly) statistične značilnosti nismo dosegli, najverjetneje zaradi variabilnosti med biološkimi ponovitvami. S primerjalno analizo kontrolnih različic smo potrdili ustreznost izbrane metode za funkcionalno analizo različic gena EGLN1. Zmanjšana stabilnost genetskih različic nakazuje njihov vpliv na funkcijo proteina. S tem smo potrdili, da nova različica p.(Pro358Ser) potencialno vpliva na razvoj ECYT.

• Language: Slovenian
• Keywords: družinska eritrocitoza, gen EGLN1, različica gena, protein PHD2, stabilnost proteinov
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2025
• PID: 20.500.12556/RUL-171968
• COBISS.SI-ID: 247857155
• Publication date in RUL: 05.09.2025

Secondary language

• Language: English
• Title: Effect of variants in the EGLN1 gene on the stability of the protein product

Abstract

Erythrocytosis (ECYT) is a pathological condition characterized by an increased number of red blood cells and consequently higher concentrations of hemoglobin in the blood. A key role in regulating erythropoiesis is played by the hypoxia-inducible factor (HIF), whose stability and activity are regulated by prolyl hydroxylase 2 (PHD2), encoded by the EGLN1 gene. Certain genetic variants of EGLN1 reduce stability of EGLN1/PHD2 and are therefore associated with the development of erythrocytosis. The aim of this master’s thesis was to investigate the effect of two new genetic variants in the EGLN1 gene, c.1072C>T, p.(Pro358Ser) and c.1124A>G, p.(Glu375Gly), on the stability of the PHD2 protein. The stability of the EGLN1/PHD2 variants (p.(Glu375Gly) and p.(Pro358Ser)) was compared with the wild type (WT) as well as with a positive (p.(His374Arg)) and negative (p.(Gln157His)) control. The selected EGLN1 variants and controls were introduced into the HEK293 cell line, and after 24 hours, translation was halted by the addition of cycloheximide. Cells were lysed at various time points (T0, T3, T6, T10) following cycloheximide treatment, and protein expression was assessed via western blot analysis. The results showed that the WT and negative control did not affect EGLN1/PHD2 protein stability, whereas the other variants led to reduced stability. Statistically significant results were obtained for the p.(His374Arg) and p.(Pro358Ser) variants, while statistical significance was not achieved for p.(Glu375Gly), likely due to biological variability between replicates. Comparative analysis of the control variants confirmed the suitability of the selected method for functional analysis of EGLN1 gene variants. Reduced protein stability of the tested genetic variants indicates an impact on protein function, confirming that the new variant p.(Pro358Ser) potentially contributes to the development of ECYT.

• Keywords: familial erythrocytosis, EGLN1 gene, gene variant, PHD2 protein, protein stability